Studies on asexual stage immunity to P. falciparum 1.) Evaluate the merozoite antigen PfRH5 as a vaccine candidate. Our collaborators at Oxford University (Dr. Simon Draper et al.) are pursuing this protein as a vaccine candidate and we have contributed to various pre-clinical studies using our standardized growth-inhibition assay (GIA). One clinical trial using immunization with recombinant viruses has already been conducted but the immunogenicity was not sufficient to justify a parasite challenge. However, a second clinical trial using recombinant protein is in progress and our immunogenicity results to date will likely support a challenge infection late in 2017. 2.)In addition, we are initiating a more detailed study of immune responses in field sites to PfRH5 as well as other molecules associated as part of an invasion complex with this molecule - PfCyRPA, PfRIPr, etc. 3.) Investigate other targets of merozoite immunity - AMA1 While AMA1 is a prominent vaccine candidate, it has two issues:1) antigenic polymorphism so that antibody responses tend to be strain-specific and 2) lack of protection in human clinical trials. We have collaborated with LMVR investigators (Dr. Louis Miller) to show that using AMA1 in conjunction with its partner protein RON2 produces antibodies with greater activity in the GIA assay. This complex is required for triggering junction formation between the merozoite and the red cell surface. 4.) Studies of immunity to malaria in Kenieroba, Mali. Our 4-year investigation of the acquisition of immunity to malaria in Malian children represents perhaps the most detailed longitudinal study of malaria in African children that has been conducted. Antisera collected in this 4-year study continue to be used to evaluate other blood-stage vaccine candidates. For example, with Dr. Takafumi Tsuboi we have identified LSA-3 as a possible vaccine candidate based on antibody responses to a large array of blood-stage proteins produced in a wheat germ expression system. 5.) We continue to work with Drs. Amy Bei and Dyann Wirth (Harvard University), who are studying changes in frequency of different P.falciparum clones over time in Senegal. We have collaborated in examining the influence of human immune responses on the changes in clonal parasite patterns, particularly related to the observation of parasites with common genetic signatures (CGS). Initial studies addressed the possibility that the human antibody responses affected the selection and persistence of parasite clones with CGS. More advanced studies are in progress. 6.) We are also collaborating with Oxford University on a major EU-funded project to develop a 3-stage multi-component vaccine for P. falciparum. Studies on parasite sexual stages and transmission blocking vaccine candidates: 1.) Develop quantitative methodology for analysis of the standard mosquito membrane feeding assay (SMFA) to evaluate transmission blocking activity. The gold standard assay to evaluate the ability of antibodies to block transmission to mosquitoes is the SMFA, and we have performed an in depth study of this assay to define its characteristics in order to have confidence in assessing potential transmission blocking vaccine (TBV) candidates. We have shown that the SMFA is quite reproducible at high concentrations of antibody but highly variable at low concentrations, and we have worked with the Biostatistics group at NIAID to develop a statistical model of the assay. Recently we have tested a modified model of the assay to show for the first time that we can predict the impact of a specific antibody on the reduction of P. falciparum parasite prevalence in mosquitoes based on the reduction in oocysts in an SMFA and the number of oocysts in control mosquitoes. Reduction in malaria prevalence in mosquitoes by an antibody is key to reducing transmission in the field. 2.) Search for and evaluate new possible transmission blocking vaccine candidates. Using SMFA we have evaluated a number of potential transmission blocking vaccine candidates. In collaboration with colleagues at Oxford University we have produced and tested a number of known and unknown sexual stage proteins from P. falciparum. Sera from immunized mice have been evaluated by SMFA but none have shown high levels of oocyst inhibition. Further we have tested antibodies produced by collaborators to large numbers of other sexual stage and mosquito vaccine candidates to compare their activity. 3.) Construction and evaluation of human/humanized monoclonal antibodies (mabs) to several sexual stage parasite proteins. We are currently comparing a number of mabs to Pfs25 and other candidates as a foundation for human clinical trials. Passive administration of these antibodies will allow evaluation of transmission blocking in vivo and might eventually be used in elimination campaigns. We are comparing them in SMFA, mapping their epitopes, and determining their binding properties. 4.) We have initiated studies to examine the differentiation pattern of P. falciparum gametocytes in culture using RNASeq. We have obtained data on RNA transcription during 2- weeks of differentiation and we are analyzing the data to identify better markers of early and late stage gametocyte development. 5.)In addition, we are conducting a series of experiments to determine what serum-derived factors are necessary for successful differentiation of gametocytes so that robust numbers of oocysts can be obtained after mosquito infection. Mass spectrometry of good and bad sera have shown consistent differences between them (M Llinas collaboration). Field studies of malaria transmission in Mali 1.) We have assessed the presence of asexual and sexual stage parasites in residents of Kenieroba, Mali throughout the year. In the spring of 2013 we initiated a new study (NIH 13-I-N107) to address the limited information on transmission in malaria endemic areas, and in 2014 we completed the field aspect of this study. Volunteers representing all age groups were finger pricked twice per month for 1 year to collect DNA and RNA. We previously completed analysis of over 10,000 samples of parasite DNA on filter paper. In November, 2013 (wet season), P. falciparum prevalence in the cohort was 37.0%, and in May of 2014 (dry season), the prevalence dropped to 10.0%. We also analyzed the longitudinal prevalence of the cohort; we observed that the 9-16 years old age group had the highest median longitudinal prevalence compared to the other age groups, and males had a higher median than females (22.0%). 2.)We showed for the first time that increasing P.falciparum longitudinal prevalence throughout the year was associated with decreasing risk of clinical malaria. This suggests that those with persistent parasite carriage acquire stronger protective immunity against clinical malaria. 3.) We added a new barcoding procedure based on a methodology developed at Harvard University to determine how many different clones of parasites an individual was carrying. This allowed us to show that at least 70% of infections were polygenomic, so that most people in the population are carrying more than one parasite clone at any given time. Moreover, this distribution held during both the wet and dry seasons of the year. 4.) Using the RNA collections from the same study, we used a qRT-PCR procedure to identify Pfs25 mRNA specific for gametocytes. We completed RNA analysis to determine who is carrying gametocytes. Interestingly, even though they may not be visible by microscopy, more than 80% of people with parasites also have detectable gametocytes by molecular criteria. This shows that no one group can be uniquely targeted for interventions to reduce transmission.